Prostate cancer (PCa) is the most common male urological malignancy, and it is not curable at its advanced stages. It is proposed that a rare population of functionally distinct cancer cells possess the extensive proliferative and self-renewal potential necessary to create a tumor;these are the cancer stem cells (CSCs). Progress in identification of CSCs in solid tumors including those in the breast and brain has prompted strong belief that PCa is a stem cell disease. However, definitive evidence of the existence of CSCs in PCa is lacking. Our long-term objective is to identify, isolate, and characterize a pure population of CSCs from PCa. To achieve this goal, we will first identify certain phenotypic features of cancer cells that co-segregate with functional attributes of sternness, most importantly self-renewal. We will stratify cells using fluorescenceactivated cell sorting based on three criteria: 1) the expression of cell surface markers that have been shown to be expressed by normal prostate stem cells or CSCs in other tissues;2) the ability to actively efflux a fluorescent dye, Hoechst 33342, a characteristic of stem cells in other tissues;and 3) the expression of genes involved in stem cell renewal including Wnt and Hedgehog pathways using reporter genes. We will then test the relative ability of cell populations with or without these properties to form tumors in immunodeficient mice. Methods to grow and expand enriched "stem" populations will be developed. Finally, we will identify new markers for CSCs by comparing gene expression profiles of cell populations enriched for CSCs to more differentiated cells and generating antibody libraries against cell surface antigens on enriched "stem" populations. Relevance: Cancer stem cell theory suggests that only a minority of cells, the cancer stem cells (CSCs), within a tumor are truly malignant and capable of driving tumor growth and metastasis, whereas the bulk of a tumor is actually made up of differentiated non-tumorigenic cells. Standard therapies shrink tumors by eradicating the sensitive non-tumorgenic cells, but the tumors recur because the resistant stem cells repopulate the cancer. Our results will help the design of new therapies targeting prostate cancer CSCs that effectively kill the CSCs, rendering the tumors unable to maintain themselves or grow, thus effecting a cure.